Enterohemorrhagic Escherichia coli (EHEC) strains are important human food-borne pathogens (Kaper et al., 2004. Nat Rev Microbiol 2:123-140). The clinical manifestations of EHEC infections range from watery diarrhea, or hemorrhagic colitis (HC), to the most severe outcome, the life-threatening hemolytic-uremic syndrome (HUS) (Karmali 1989. Clin Microbiol Rev 2:15-38). The infection correlates with ingestion of contaminated meat or vegetables, but is also transmitted by water or even person-to-person contact (Caprioli et al., 2005. Vet Res 36:289-311; Griffin and Tauxe, 1991. Epidemiol Rev 13:60-98). Sporadic or massive outbreaks have been reported in several developed countries. In other countries, such as in Argentina, HUS shows an endemic behavior and represents a serious public health problem with high morbidity and mortality values (Lopez et al., 2000. Infect Dis Clin North Am 14:41-65, viii; Rivas et al., 2006. Medicina (B Aires) 66 Suppl 3:27-32).
A striking feature of EHEC infection is the production of potent Shiga toxins, responsible of HUS development (Noel and Boedeker, 1997. Dig Dis 15:67-91; O'brien et al., Curr Top Microbiol Immunol 180:65-94). The Shiga toxin family is a group of structurally and functionally related AB5 exotoxins, which includes Shiga toxin (Stx) produced by Shigella dysenteriae serotype 1 and the Shiga toxins that are produced by enterohemorrhagic Escherichia coli (EHEC) strains. The EHEC can produce two types of Stx, Shiga toxin type 1 (Stx1) and type 2 (Stx2), and their allelic variants. The genes for Shiga toxins are encoded by lysogenic lamboid bacteriophages (Schmidt, 2001. Res Microbiol 152 (8): 687-95), and a given bacteria may express more than one Stx, as they may contain more than one Stx-encoding bacteriophage.
All Shiga toxin family members have an AB5 molecular configuration (Stein et al., 1992. Nature 355 (6362): 748-50; Fraser et al., 1994. Nat Struct Biol 1 (1): 59-64), in which an enzymatically active monomeric A subunit, StxA (which has a molecular mass of 32 kDa) is non-covalently associated with a B subunit, StxB, responsible for binding to cell surface receptors. StxB is a homopentameric protein (a pentamer of identical monomers, each having a molecular mass of 7.7 kDa). StxB forms a ring-like structure with a central pore into which the carboxyl terminus of StxA inserts (Fraser et al., 1994. Nat Struct Biol 1 (1): 59-64). StxA and the StxB subunits are secreted into the bacterial periplasm, where they assemble non-covalently into the holotoxin, as was initially described for heat-labile enterotoxins from E. coli (Hirst et al., 1984. Proc Natl Acad Sci USA 81 (24): 7752-6).
StxA possesses a highly specific RNA N-glycosidase activity that cleaves an adenine base at position 4,324 on the α-sarcin loop located on domain VI of 28S ribosomal RNA (rRNA) of eukaryotic ribosomes, thereby inhibiting elongation factor-dependent aminoacyl tRNA binding and subsequent chain elongation (Endo et al., 1988. Eur J Biochem 171 (1-2): 45-50). Bacterial ribosomes are also a substrate for StxA, and exposure to Shiga toxin type 1 (Stx1) results in decreased proliferation of susceptible bacteria (Suh et al., 1998. Biochemistry 37 (26): 9394-8).
StxA subunit is composed of two fragments linked by a disulfide bridge, which is proteolytically cleaved by enzymes in the cytosol and endoplasmic reticulum, generating the A1 subunit of 27 kDa responsible for the enzymatic activity and releasing the smaller A2 fragment (Garred et al., 1995. J Biol Chem 270 (18): 10817-21). Austin and collaborators showed that while StxA and StxB can form the holotoxin spontaneously in vitro, the recombinant A1 fragment cannot bind to StxB, indicating that the fragment A2 is essential for the assembly of the holotoxin (Austin et al., 1994. Infect Immun 62 (5): 1768-75).
While StxA is responsible for the toxic effect, the StxB pentamer is responsible for binding to a specific receptor in eukaryotic cells. StxB binds to the neutral glycosphingolipid globotriaosylceramide (Gb3; also known as Cd77 or the Pk blood group antigen), which is present on the surface of cells (Jacewicz et al., 1986. J Exp Med 163 (6): 1391-404; Lindberg et al., 1987. J Biol Chem 262 (4): 1779-85; Waddell et al., 1990. Proc Natl Acad Sci USA 87 (20): 7898-901), leading to subsequent internalization of the toxin. In the absence of StxA, StxB still adopts a pentameric structure that is functionally equivalent to the holotoxin in its ability to bind the receptor (Donohue-Rolfe et al., 1989. Mol Microbiol 3 (9): 1231-6). Each B subunit monomer comprises two three-stranded antiparallel β-sheets and an α-helix. The pentamer forms a ring-like structure with a central pore of about an 11 Å diameter delimited by five α-helices and surrounded by β-sheets from pairs of adjacent monomers, forming six-stranded antiparallel β-sheets (Stein et al., 1992. Nature 355 (6362): 748-50).
The resolution of the crystal structure of subunit B of Shiga toxin 1 (Stx1B) complexed with a receptor Gb3 trisaccharide analogue revealed three potential binding sites within each monomer of B subunit (referred to as sites 1, 2 and 3) (Ling et al., 1998. Biochemistry 37 (7): 1777-88). Therefore, there are 15 Gb3 binding sites in the StxB pentamer. All 15 of the Gb3-binding sites face in the same direction, distal to the A subunit, thereby identifying the membrane interaction surface within the pentamer. The Gb3-binding sites do not interact with each other either directly or through conformational changes.
Site 1 is located in a groove between adjacent B subunits and is characterized by a hydrophobic interaction between the phenyl ring of Phe-30 and the Galβ of the receptor and by hydrogen bonds involving Asp-17, Thr-21, Glu-28 and Gly-60. Site 2 is located on the opposite side of the phenyl ring of Phe-30 in a crevice defined by Gly-63, Asn-32, Arg-33 and Ala-56. The third binding site involves hydrophobic stacking interactions of Galβ against the indole ring of Trp-34 (located in the α-helices surrounding the central pore of Stx1B) and a hydrophobic interaction between Galα and Trp-34 of the adjacent monomer. In addition, Galα hydrogen bonds to Trp34 and Asn35 as well as Asp18 from an adjacent monomer. From these results it can be concluded that at least sites 1 and 3 require the correct assembly of the pentamer to be functional. Mutational studies with Stx1B demonstrated that sites 1 and 2 mediate high affinity interactions and that Stx1 cytotoxic activity is mediated primarily by Gb3 binding to sites 1 and 2. However, site 3 mediates low affinity interactions (Bast et al., 1999. Mol Microbiol 32 (5): 953-60). Although the affinity of site 3 may be too low to significantly contribute to the strength of cell binding, Ling and collaborators proposed that it could serve other purposes. One reason for this belief is the fact that the tryptophan residue at position 34 is conserved despite being fully exposed to the solvent. These authors proposed that site 3 may play a role helping to sequester a greater number of Gb3 molecules in the membrane below the toxin (Ling et al., 2000. Structure 8 (3): 253-64). Thus, all three oligosaccharide-binding sites are required for full biological activity.
The study of Stx2B has been hampered due to the difficulty of expressing large quantities of the biologically active form (Acheson et al., 1995. Infect Immun 63 (1): 301-8). However, analysis of the crystallographic structure of Stx2 predicted the presence of the corresponding trisaccharide binding sites on its B subunit, but also demonstrated that the conformation at site 2 differs distinctively from that of the Shiga toxin from Shigella and Stx1 B subunits (Fraser et al., 2004. J Biol Chem 279 (26): 27511-7). However, the residues involved in sugar-binding are either conserved in the Shiga toxin family, or are conservatively substituted (Ling et al., 2000. Structure 8 (3): 253-64). One exception is the substitution of Glu16 in site 2 of Stx2B for aspartic acid in Stx1B, where the water-mediated hydrogen bond seen for Glu16 is replaced by a direct interaction for Asp16. This substitution might be responsible in part, for the reduced Gb3 affinity of Shiga toxin 2 and its variants.
Site-directed mutagenesis has shown that the conserved residue Gly60 is essential for the cytotoxicity of Stx1 and Stx2. Substitution of this residue would alter the conformation of the β5-β6 loop, which is involved not only in site 1 but also in site 2 (Perera et al., 1991. J Bacteriol 173 (3): 1151-60). Site-directed mutagenesis has also shown that Arg33 plays an important role in the cytotoxicity of Stx1 and Stx2. The results can be explained by the extensive involvement of its side chain in hydrogen bonding to the terminal galactose of Gb3 trisaccharides in site 2 (Ling et al., 2000. Structure 8 (3): 253-64).
The prototype of the Shiga toxin family is the Stx produced by Shigella dysenteriae, which is almost identical to the Stx1 produced by E. coli, differing in a single amino acid in the catalytic subunit (O'Loughlin and Robins-Browne, 2001. Microbes Infect 3 (6): 493-507). Both Stx1 and Stx2 have different variants, with Stx2 being the most diverse. The Stx1 family consists of Stx1 and Stx1c while Stx2 contains the variants Stx2c, Stx2c2, Stx2d, Stx2dactivable, Stx2e and Stx2f. Stx1 and Stx2 only have a 56% identity to the amino acid sequence level (Jackson et al., 1987. Microb Pathog 2 (2): 147-53). Stx2 variants are 84-99% homologous to Stx2.
While there are significant similarities between Stx1 and Stx2 in their basic structure, receptor recognition and biochemical mechanisms of action, there are considerable differences in the clinical impact of patients infected with EHEC strains producing Stx1, Stx2 or both. In principle, one would predict that both Stx1 and Stx2 would put the patient at risk of developing HUS. However, numerous epidemiological studies have shown that Stx2-producing strains are more frequently associated with HUS development than Stx1-producing strains or strains that produce both toxins (Scotland et al., 1987. Epidemiol Infect 99 (3): 613-24; Ostroff et al., 1989. J Infect Dis 160 (6): 994-8; Donohue-Rolfe et al., 2000. J Infect Dis 181 (5): 1825-9).
Regarding the differences between Stx1 and Stx2, the most significant of them are the differences in affinities for the Gb3 receptor and ability to induce toxicity. Although Stx1 and Stx2 have the same functional receptor, Stx1 has a 10 fold higher affinity for Gb3 compared to Stx2 (Head et al., 1991. Biol Chem 266 (6): 3617-21). A study using the BIAcore system showed that, although the rate of association of Stx1 to Gb3 is greater than Stx2, the dissociation of Stx2 from the receptor is slower than Stx1 indicating that, while Stx2 binds to the receptor slower, it also dissociates at a slower rate (Nakajima et al., 2001. J Biol Chem 276 (46): 42915-22).
Despite the higher affinity of Stx1 for Gb3, and consistent with the epidemiological studies, Stx2 has a lethal dose in mice of 50% (LD50), 400 times lower than Stx1 (Tesh et al., 1993. Infect Immun 61 (8): 3392-402). Similar results were also obtained when human renal microvascular endothelial cells were treated with Stx1 or Stx2, with Stx2 being found to be about 1000-fold more toxic (Louise and Obrig, 1995. J Infect Dis 172 (5): 1397-401). Different studies have made it clear that the B subunits are critical determinants of the differential toxicity of Stx1 and Stx2 in vivo. In cell-free in vitro translation inhibition assays, Stx1 and Stx2 A subunit toxicities are indistinguishable, suggesting that the enzymatic activities of these subunits are not responsible for the large in vivo differences between the two toxins. In contrast, animal models comparing wild-type and chimeric Stx toxicity demonstrate that the presence of the Stx2B subunit is a critical determinant of lethality in vitro and in vivo (Weinstein et al., 1989. Infect Immun 57 (12): 3743-50; Head et al., 1991. Biol Chem 266 (6): 3617-21; Lingwood, 1996. Trends Microbiol 4 (4): 147-53; Marcato et al., 2003. Infect Immun 71 (10): 6075-8).
Using mass spectrometry techniques, Kitova et al. reported that Stx1B was primarily pentameric at subunit concentrations ranging from 5 to 85 μM, independently of the ionic strength (Kitova et al., 2005. J Am Soc Mass Spectrom 16 (12): 1957-68; Kitova et al., 2009. Biochemistry 48 (23): 5365-74). These data were supported by circular dichroism (CD) and dynamic scanning calorimetry (DSC) studies of Stx1B showing a highly thermostable pentamer (Pina et al., 2003. Biochemistry 42 (31): 9498-506). In contrast, the degree of assembly of Stx2B subunit is strongly dependent on temperature, subunit concentration and ionic strength. At a subunit concentration of more than 50 μM, the Stx2B subunit exists predominantly as a pentamer, although smaller multimers (dimer, trimer and tetramer) are also evident. At lower concentrations, the Stx2B subunit exists predominantly as monomers and dimers. Conrady and collaborators confirmed and quantified the differences observed by Kitova et al. (2005) in a direct solution-state technique. These authors reported that the Stx2B pentamer is approximately 50 times less stable in solution at pH 7.4 than the Stx1B pentamer (Conrady et al., 2010. PLoS One 5 (12): e15153).
Another important difference between Stx1 and Stx2 is observed at the immunological level. Although these two types of Stx were initially identified because antibodies directed against one variant did not cross-react against the other, the study of cross-reactivity (and cross-neutralization) between these two toxins has been the subject of controversy over the years. In vitro studies indicate that these two toxins are serologically distinct and that antibodies directed against one toxin are not capable of neutralizing the other (Scotland et al., 1985. Lancet 2 (8460): 885-6; Karmali et al., 1986. Lancet 1 (8473): 164-5; Strockbine et al., 1986. Infect Immun 53 (1): 135-40). Similarly, Wen et al. showed that immunization of mice with genetically inactivated Stx1 or Stx2 toxoids was able to generate anti-toxin serum IgG against homologous but not heterologous toxin and that these antibodies provided specific protective immunity against challenge with only the homologous toxin (Wen et al., 2006. Vaccine 24 (8): 1142-8). However, in other studies with animals immunized with toxoid preparations of Stx1 or Stx2, animals showed cross-protection against challenge with either toxin due to the production of antibodies against the A subunit. Bielaszweska et al. found that immunization of rabbits with chemically inactivated Stx1 or Stx2 toxoids, or the A subunits of each toxin, prevented the heterologous toxin localization in target tissues during development of the systemic pathology mediated by the toxin. In that study, the B subunits of Stx1 or Stx2 did not provide heterologous protection (Bielaszewska et al., 1997. Infect Immun 65 (7): 2509-16). Further evidence of cross-protection in vivo was reported by Ludwig et al., where protection of rabbits against challenge with Stx1 was obtained by immunization with a chemically inactivated Stx2 toxoid (Ludwig et al., 2002. Can J Microbiol 48 (1): 99-103).
Cross-reactivity between B subunits of Stx is also controversial. Some studies have shown that antibodies against the B subunits of Stx1 or Stx2 do not provide cross-protection (Wadolkowski et al., 1990. Infect Immun 58 (12): 3959-65; Bielaszewska et al., 1997. Infect Immun 65 (7): 2509-16). For this reason, several authors have proposed the use of fusion proteins between Stx2B and Stx1B to provide protection against both toxins (Gao et al., 2009. Vaccine 27 (14): 2070-6; Zhang et al., 2011. Vaccine 29 (22): 3923-9). However, Tsuji et al. reported that intranasal immunization of mice with the construction Stx2B-HIS (Stx2B with a histidine tag) was able to confer cross-protection against Stx1 but that the opposite was not true (Tsuji et al., 2008. Vaccine 26 (17): 2092-9).
Stx2 variants are distinguishable by differences in biological activity, immunological reactivity or receptor specificity. Stx2 and its variants preferentially bind to Gb3 glycosphingolipid, with the exception of Stx2e, which binds preferentially to globotetraosylceramide (Gb4) (Lingwood, 1996. Trends Microbiol 4 (4): 147-53). While Stx2 and Stx2c are the most virulent Stx2 variants, and are associated with the majority of cases of HUS (Boerlin et al., 1999. J Clin Microbiol 37 (3): 497-503), Stx2e is the only one not associated with hemorrhagic colitis and HUS in humans and is instead responsible for the edema disease in pigs (MacLeod et al., 1991. Vet Pathol 28 (1): 66-73). Furthermore, Stx2dactivable differs from all other types of Stx in that it can be activated by elastase, a component of intestinal mucus which cleaves the last two residues of the A subunit to release the fragment A2 (Scheiring et al., 2008. Pediatr Nephrol 23 (10): 1749-60).
Despite the magnitude of the social and economic problems caused by EHEC infections, no licensed vaccine or effective therapy is presently available for human use. One of the biggest challenges is to develop an effective and safe immunogen to ensure non-toxicity but also a strong input to host immune system to induce long-lasting, high affinity antibodies that ensure a good neutralization capacity in serum. The B subunit of Stx2 (Stx2B) is the most attractive candidate because, among the Stx family, Stx2 is the most pathogenic toxin, and a Stx2B-based immunogen would protect against the Stx most related to HUS development (Smith et al., 2006. Vaccine 24:4122-4129; Wen et al., 2006. Vaccine 24 (8): 1142-8; Tsuji et al., 2008. Vaccine 26 (17): 2092-9). In addition, the B subunit represents the binding unit of the toxin and is non-toxic for mammalian cells (Donohue-Rolfe et al., 1991. Rev Infect Dis 13 Suppl 4:S293-297; Lingwood, 1996. Trends Microbiol 4 (4): 147-53). Antibodies able to block the binding process to the specific receptor (Gb3) in mammalian cells should prevent the first step of the toxicity cascade (Ling et al., 1998. Biochemistry 37 (7): 1777-88). In addition, an Stx-based vaccine against HUS would not only protect against known EHEC strains, typically O157 and non-O157 serotypes, but it would also be useful against new or rare pathogenic strains of Shiga-toxin-producing E. coli, as was the case of the recent large outbreak of HUS caused by the 0104:H4 strain (Beutin et al., 2012. J Virol 86:10444-10455; Scheutz et al., 2011. Euro Surveill 16).
Despite multiple approaches, a successful Stx2B-based immunogen has not been obtained, mainly because Stx2B is a very poor immunogen (Marcato et al., 2001. J Infect Dis 183:435-443; Imai et al., 2004. Infect Immun 72 (2):889-895). Two of the three binding sites are formed by residues contributed by neighboring monomers, thus requiring the right assembly of the pentamer for the binding sites to be active (Ling et al., 1998. Biochemistry 37 (7): 1777-88). As discussed above, the Stx2B pentamer is only marginally stable in the absence of the A subunit, and when used as immunogen, it is unable to raise specific antibodies against conformational epitopes that are located mostly at the interfaces between monomers of the pentamer.